Successful progression through spermatogenesis is crucial for normal gamete formation and for transferring the genetic information to the next generation. Unfortunately, in humans, infertility affects approximately 15% of couples worldwide and the male partner is responsible for the infertility in at least half of all cases. Sumoylation has emerged as a critical regulatory event in cell function and has been implicated in various diseases including cancer. SUMO proteins have recently been localized to specific subdomains of germ and somatic testicular cells and the obtained evidence implicated sumoylation in different aspects of normal and impaired spermatogenesis. However, unlike somatic cells, in which numerous sumoylated proteins have been identified and studied, targets and regulation of SUMO in the testis are mostly uncharacterized. The objective of this study is to identify and initially characterize specific targets of sumoylation in mitotic spermatogonia, meiotic spermatocytes, differentiating spermatids, and testicular somatic cells. Populations enriched for specific cell types will be obtained from both pubertal mice, undergoing their initial wave of spermatogenesis, and adult mouse testis. Highly validated immunoprecipitation procedures using anti-SUMO antibodies will be followed by liquid chromatography-mass spectrometry protein identification and bioinformatic analysis. Spermatogonia-related experiments using freshly isolated cells will be supplemented and extended using cell lines and stable isotope labeling with amino acids in cell culture (SILAC) to study changes in the sumoylated proteomes of spermatogonia as they undergo differentiation. The results obtained from different experimental settings will be further confirmed using co-immunoprecipitation and localization studies. Together, the proposed research will advance knowledge across the field of germ cell biology by elucidating the new protein networks and regulatory pathways that are necessary for progression through spermatogenesis. PUBLIC HEALTH RELEVANCE: In at least half of all cases of human infertility (one in every six couples who are trying to conceive) male spermatogenic failure is a major or contributing cause. This work focuses on studies of the biological functions of novel proteins (SUMO) at the level of individual targets and corresponding pathways, leading to the better understanding of possible causes of male infertility and development of novel safe contraceptives, thus improving human healthcare.